Inhibition of leukemia utilizing 5-methyltetrahydrohomofolate

ABSTRACT

A method of inhibiting methotrexate sensitive leukemia L1210 and methotrexate resistant leukemias derived from L1210, such as variants L1210/FR-8, L1210/M-46-R, L1210/C-95, and L1210/M-66-3A in mice. This method produces optimum results as to the resistant leukemias as well as significant activity as to the sensitive leukemia. The compound employed is 5-methyltetrahydrohomofolate utilized as an injectable in a dosage regiment of 12.5-1600 mg/kg/inj/diem. The mechanism of action prefers dosages for at least two to four days, and in mice optimum results are obtained when treatment is commenced on day 1 following leukemia innoculation and is continued to the death of mice.

United States Patent [1 1 Mead [4 1 Dec. 24, 1974 INHIBITION OF LEUKEMIAUTILIZING 5-METHYLTETRAHYDROHOMOFOLATE [75] Inventor: John A. R. Mead,Bethesda, Md.

[73] Assignee: The United States of America as represented by theSecretary of the Department of Health, Education and Welfare,Washington, DC

[22] Filed: July 24, 1972 21 Appl. No.: 274,749

[52] US. Cl. 424/251 [51] Int. Cl... A6lk 27/00 [58] Field of Search424/251 [56] References Cited OTHER PUBLICATIONS Chemical Abstracts 60:9277(h) (1964). Chemical Abstracts 66: 7270.7w (1967).

Chemical Abstracts 71: 6726lv (1969).

Primary ExaminerJ erome D. Goldberg [57] ABSTRACT A method of inhibitingmethotrexate sensitive leukemia Ll2l0 and methotrexate resistantleukemias derived from Ll210, such as variants Ll2lO/FR-8, L1210/M-46-R,Ll2l0/C-95, and Ll2lO/M-66-3A in mice. This method produces optimumresults as to the resistant leukemias as well as significant activity asto the sensitive leukemia. The compound employed is5-methyltetrahydrohomofolate utilized as an injectable in a dosageregiment of 125-1600 mg/kg/inj/diem. The mechanism of action prefersdosages for at least two to four days, and in mice optimum results areobtained when treatment is commenced on day 1 following leukemiainnoculation and is continued to the death of mice.

3 Claims, No Drawings INHIBITION OF LEUKEMIA UTILIZING-METHYLTETRAHYDROHOMOFOLATE The present application contains subjectmatter related to Ser. No. 263,559, filed June 16, 1972, Knott et al.,assigned also to U.S.A.-HEW, entitled Synthesis of N-Methyltetrahydrohomofolic Acid and Related Reduced Derivatives ofl-lomofolic Acid, which deals with the preparation of the presentcompound per se and is incorporated herewith by reference.

In precise form, the related Knott et al. application commences withhomofolic acid (HFA) which is reduced with hydrogen and platinum oxideto tetrahydrohomofolate (Tl-Il-IF). This latter material is then reactedwith formaldehyde to produce N N -methylene TI-Il-IF which is thenreduced with sodium borohydride to produce the desired N -methyl Tl-IHFin the disodium salt form. The production of the related folatederivatives (i.e., N -methyltetrahydrofolate) has been described in theliterature as follows:

W. Sakami, Biochem. Prep., 10, 103 (1963) V. S. Gupta and F. M.l-Iuennekens, Arch. Biochem.

Biophys., 120, 7l27l8 (1967) Additionally, in the patent art theproduction of tetrahydrohomofolic acid salts is described in:

U.S. Pat. No. 3,468,886 Mosher et al. (USAHEW) U.S. Pat. No. 3,637,695Kim et al. (USA-HEW) In modern leukemia therapy, the standard drug ofcomparison for several years has been methotrexate (amethopterin). Themodus of action in vivo of this compound in enzymatic reaction is toinhibit the action of dihydrofolate reductase and this and similar drugsconstituting 4-amino analogues of folic acid are known as the4-amino-antifolates. It has recently been realized that where leukemiais strongly resistant to the 4- amino-antifolates and is characterizedby high levels of dihydrofolate reductase, certain homofolatederivatives show a substantial antileukemic effect J. A. R. Mead, Ann.NY. Acad. Sci., 186, 514-515, Nov., 1971.

It has been shown that tetrahydrohomofolate is a potent inhibitor invitro in the enzymatic reaction of thymidylate synthetase and the enzymeis metabolically beyond or below the action of the dihydrofolatereductase. Thus, it is considered possible that dihydrohomofolate andpossibly homofolate by conversion in vivo to a specific inhibitor ofthymidylate synthesis might block the growth of amethopterin-resistantcells having high levels of dihydrofolate reductase J. A. R. Mead etal., Cancer Research, 2374-2379 (1966). In summation, whereas the knowncompounds utilized have been active at (2) in the accompanying Chart 1,the tetrahydrohomofolates are active at (3), and it is believed in thepresent invention, the 5-methyltetrahydrohomofolate (S-MeTl-IHF) isactive at (4) relative to the action of methionine synthetase.

CHART I Enzymic Reactions of Folates PteGlu; (D

PteGlu PteG1u1 (D S-CI-Ia-HdteGlu H PteGlu 5-HCO-H4PteGlu G) G) (3 G) G)There is also considerable evidence that the presence of methyl at the Nposition has retarded the chemical breakdown of H, to H by oxidation andthus S-methyl Tl-IHF is more stable than THHF. For purposes of thepresent specification and claims, this compound, 5- methyl Tl-Il-IF, bydefinition includes the free acid, mono and dialkali metal salts, andpharmaceutically acceptable acid addition salts for adjustingsolubility. Thus, the present invention is directed towards the freeacid as well as the mono and dipotassium salts, the mono and disodiumsalts, and analogous soluble salts of Group IA'of the Periodic Table,such as lithium, rubidium, and caesium. Additionally, pharmaceuticallyacceptable acid addition salts and amine salts, such as those formedfrom a strong base such as cyclohexylamine, are also included in thisdefinition.

Although not a required component of the injectable, due to the tendencyof the tetrahydrohomofolates to oxidize, it is of auxiliary value toinclude a mild reducing agent which is pharmaceutically utilizable, suchas sodium ascorbate, cysteine, mercaptoethanol, 1,2- mercaptopropanol,Clelands reagent, etc.

The usefulness of the reduced homofolate derivatives, especially inmethotrexate-resistant variants of leukemia L 1 210, has been apparentfor some time. One difficulty has been in that the tetrahydrohomofolateper se and its alkaline salts have been highly fugitive due to rapidoxidation which not only posed problems during its formation but in itsuse. The present derivative, N -methyltetrahydrohomofolate, showsgreater stability and enables its use in animal (mice) therapy.Especially this compound or drug is an effective antileukemic agentagainst methotrexate-resistant L12l0/FR-8 leukemia which contains highlevels of dihydrofolate reductase. Additionally, the S-methyl compoundhas been found active against other Ll210 variants which aremethotrexate resistant, such as L1210/M-46-R, L1210/C-95, andL1210/M-66-3A.

It is noted in the formula gpmofolic Acid COOH H CH2 chem. Pharm.,2871-2878 (1971)].

In animal studies with mice where the N -methyltetrahydrohomofolic acidwas compared with methotrexate (MTX) against L121O sensitive and theL121O resistant variants, the results showed that the S-methyl compoundwas vastly superior to MTX in increasing' life span and median survivaltime even at low dosages of 12.5 mg/kg as to the resistant tumor and asignificantly active compound against the sensitive tumor. From theselow dosages up to so-called optimum dosages of 400 mg/kg, the S-methylcompound operated in an optimum manner where the 1P treatment schedulewas daily from day 1 until death.

In the accompanying Chart ll, it was noted that the superiority of theS-MeTHHF over methotrexate is not apparent utilizing the L1210 sensitivestrain but that the S-methyl would be operable.

Additionally, Chart Ill shows vividly as to the L1210/FR-8 thesuperiority in ILS and MST over methotrexate. In the comparison chart itis noted that the dosages for methotrexate are comparatively low due tothe differential potency and toxicity of this drug.

Chart IV shows additional animal studies with mice using the disodiumsalt wherein an additional variant (namely, L12l0/M-46-R) resistant tomethotrexate was utilized. This study indicates that an increase in ILSof 75 percent resulted at the low dosage of 12.5 mg/kg/inj.

CHART 11 Schedule Dependency Studyof NSC-139490 with the L l210Sensitive and the L1210 Variant Resistant to MTX (FR-8/R Tumor) Ll210L1210 Dose Sensitive FR-8/R Compound (NSC No.) 1P Treatment (mg/kg] Med.Med. Vehicle, Physical St., pH Schedule inj) S.T. 71 ILS S.T. 7? lLS5-Methyltetrahydrohomofolic (139490) Day 1 800 13 44 13 44 until death400 67 16.5 83 200 14 55 122 100 12 33 21 5 139 50 13 44 26 189 Days 1-9800 12 33 16 78 400 14 55 18 I00 200 13.5 50 16 5 83 100 12 33 16 78 5O12 33 15 5 72 Days 1,5,9 1600 10 11 16 78 800 12 33 15 67 400 10 ll 1344 200 10.5 17 13 44 Days 1,5,9 200 13 44 18 100 Q3H 100 13 44 I5 67 5012 33 I5 5 72 12 33 14 55 MTX (740) Days 1-9 3.0 15.5 72 9 0 2% NaHCOso1n.pl-1 8.5 1.5 13.5 9 0 0.75 12 33 9 0 0.375 12 33 9 0 UntreatedControls 9 9 Mice: CDF,, male. 19-26 gms, 9 weeks. SC tumor 1? treatmentas indicated CHART 111 Effect of 5-Methyltetrahydrohomofolate onSurvival Time of Mice Bearing Methotrexate-sensitive (L1210) and -resistant (L1210-FR8) Leukemia 1.1210 L1210-FR8 Drug Daily Dose Median Rangeof %1LS Median Range of %ILS mg/kg(ip) survival individual survivalindividual time(days) mortalities time(days) mortalities Vehicle Control9 9-11 0 9 8-9 0 Methotrexate 3 17 13-23 88 8, 8 -12 NSC 740 1.5 2113-23 133 8 8 12 0.75 16 14-20 77 8.5 8-9 5 0.37 13 12-14 44 9 9-10 0S-Methyltetrahydro- 400 13 12-14 44 17 14-23 88 homofolate 200 14 13-1517 14-23 88 NSC 139,490 11 9-12 22 15 1.2-19 66 Tetrahydrohomofolate 40010 6-14 1 l 13 1 1-19 44 NSC 89473 200 12 lO-12 33 12 10-12 33 100 119-12 22 19 10-25 "Treatment was started on day 3 after tumortransplantation and continued daily until death. 8 mice were included ineach group.

CHART IV Effectiveness -Methyl H HF M-95l against 1.1210 FR-S and M46-Rvariants resistant to MTX MICE: CDF. male. -27 grams l0 mice/per groupVehicles:

NSC-139490 in 0.6% ascorbic acid and 2% NaHCO, pH 7.0 NSC-740 in 2%NaHCO solution. pH 8.5

SC tumor LlZl0 FR-8 SC tumor L121!) M-46-R Treatment:

1? daily from day 1 until median day of death The embodiments of thisinvention in which an exclusive property or privilege is claimed aredefined as follows:

l. A method of inhibiting Ll2l0 leukemia in mice which consists ofinjecting in said mice an effective inhibition dosage of5-methyltetrahydrohomofolate.

2. A method of treating murine leukemia selected from the groupconsisting of U210 and methotrexate resistant variants thereof in micewhich comprises injecting in said mice about 12.5-1,600 mg/kg/inj/dayfor at least 2 days with 5-methyltetrahydrohomofolate.

3. The method according to claim 2 which comprises treating methotrexateresistant variant of leukemias 0f L12l0.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO.3,856,959

DATED December 24, 1974 INVENTOR(S) John A. R. Mead, Roger L. Knott, and

Alison Ta ton-Ri b It rs certlfl ed that error appear s fil the aboveidrrtified patent and that said Letters Patent are hereby corrected asshown below:

At page 1, under Inventors, in addition to John A, R. Mead,

insert -Roger L. .Knott and Alison Taunton-Rigby. This correction isconsonant with Patent Office Paper No. 9.

Signed and sealed this 13th day of May 1975.

(SEAL) Arrest:

C. MARSHALL DANN RUTH C. MASON Commissioner of Patents Attesting Officerand Trademarks

1. A METHOD OF INHIBITING L1210 LEUKEMIA IN MICE WHICH CONSISTS OF INJECTING IN SAID MICE AN EFFECTIVE INHIBITION DOSAGE OF 5-METHYLTETRAHYDROHOMOFOLATE.
 2. A method of treating murine leukemia selected from the group consisting of L1210 and methotrexate resistant variants thereof in mice which comprises injecting in said mice about 12.5-1,600 mg/kg/inj/day for at least 2 days with 5-methyltetrahydrohomofolate.
 3. The method according to claim 2 which comprises treating methotrexate resistant variant of leukemias of L1210. 